This invention relates to the field of clinical hematology and specifically to coagulation factor monitoring of human plasma.
The prothrombin time test
The prothrombin time (PT) test is one of several clinical hematology assays of plasma in which the result is based on the time required to form a fibrin clot. It provides diagnostically useful information regarding the patency or functional integrity of coagulation factors involved in the tissue factor pathway (formerly known as the extrinsic coagulation pathway; Rapaport, 1991), viz. factors I, II, III, V, VII, and X. It also is useful for monitoring patients receiving oral anticoagulant therapy. In practice, this test is most sensitive to the concentration and/or activity of coagulation factors V, VII, and X.
Plasma deficient or defective in one or more of the above-mentioned coagulation factors typically yields a PT that is &gt;20% longer than the average PT obtained from a group of normal plasma samples. Thus, the prothrombin time represents a useful means of detecting clotting factor deficiencies and monitoring the level of oral anticoagulation.
Thromboplastins
The PT test requires a "thromboplastin" to initiate the clotting reaction. A thromboplastin may be defined generally as a substance having clot-inducing properties or activity. In the specific context of a PT test, the thromboplastin contains substantial amounts of tissue factor (coagulation factor III), an essential cofactor for the activation of coagulation factor VII (Nemerson, 1988). Generation of activated factor VII initiates a cascade of additional reactions culminating in the formation of a fibrin clot. Such a clot can be detected manually by visual inspection or automatically by means of instrumentation designed to monitor solution viscosity or translucency.
In the past, many laboratories manufactured their own thromboplastins from human or animal tissues. This practice led to highly variable reagents that yielded mean normal plasma clotting times from approximately 11-20 sec. Since these locally produced thromboplastins did not contain calcium, the one-stage PT test actually was a two-step procedure performed by (1) mixing 0.1 mL of citrated normal plasma with 0.1 mL of thromboplastin, and (2)--after warming the mixture to 37.degree. C.--initiating the clotting reaction by addition of 0.1 mL of 0,025M CaCl.sub.2 (Biggs, 1976; p. 330; p. 677).
During the past 20 years commercially available thromboplastins have come into widespread use. As a consequence, the vast majority of clinical hematology laboratories have ceased making their own thromboplastins, and several modifications have been made to the classical procedure for a PT test, notably: (1) Since all commercial thromboplastins now contain calcium, the original "two-step" procedure has been supplanted by a "one-step" or true one-stage assay whereby 0.1 mL of citrated normal plasma is combined with 0.2 mL of calcium-enriched thromboplastin; and (2) since commercial thromboplastins generally are less variable and more active than classical thromboplastins, acceptable ranges for the clotting time of normal plasma have been reduced to about 10-14 sec, with mean normal prothrombin times of about 11-13 sec.
To emphasize the distinction between a thromboplastin formulated via classical methods and used in the two-step procedure versus current commercially available thromboplastins that contain calcium and are used in the one-step procedure, we shall refer to the latter as a "thromboplastin reagent."
It is this present-day definition of a one-step/one-stage PT test, and this present-day formulation of a calcium-enriched thromboplastin reagent that defines and relates specifically to the subject of this invention: A thromboplastin reagent suitable for use in the PT test.
Current specifications for the prothrombin time test
It is important and highly pertinent to the premise of the present invention that a new and useful thromboplastin reagent suitable for the PT test must meet or exceed the performance characteristics of commercially available thromboplastin reagents currently employed by clinical laboratories for this purpose. That is, (i) as described above, it should be a one-step/one-stage clot-based assay (ii) clotting times for normal plasma should range from approx. 10-14 sec, with a mean normal PT of about 11-13 sec; (iii) when compared with an international reference thromboplastin reagent against a panel of normal and coumadin-anticoagulated plasma samples, using generally accepted methodology (vide infra), a scattergram of the logarithms of clotting times should exhibit a high positive correlation; and (iv) a useful thromboplastin reagent will be stable under conditions of normal use. The foregoing characteristics and additional information regarding procedures and performance of thromboplastin reagents suitable for use in the PT test are further described in package inserts for the commercial SIMPLASTIN.RTM. thromboplastin reagent (Organon Teknika Corp., Durham, N.C.); Dade Thromboplastin C thromboplastin reagent (Baxter Healthcare Corp., Dade Division, Miami, Fla.); Ortho rabbit brain tissue thromboplastin (Ortho Diagnostics Systems, Raritan, N.J.); and THROMBOREL.RTM. S thromboplastin reagent (Behrinkerke AG, Marburg, Germany), which are incorporated herein by reference.
Methods for preparing thromboplastins
The classical method of preparing a thromboplastin for the PT test was first described by Quick et al. (1935; 1938). The Quick thromboplastin (Quick, 1938) was prepared from minced rabbit brains by a process involving the steps of (1) dehydration and partial delipidation of the minced tissue by repeated washing with acetone, followed by drying at 37.degree. C.; (2) reconstitution of the acetone powder (0.3 g) in "5 cc. of physiologic solution of sodium chloride containing 0.1 cc of sodium oxalate" (i.e., a 5 mL aqueous solution containing approx. 0.15M NaCl plus 0,002M Na.sub.2 C.sub.2 O.sub.4); (3) incubation of the suspension at 45.degree. C. for 10 min; and (4) low-speed centrifugation for 3 min to remove large particles. Thromboplastin activity remains in the "milky supernatant liquid."
The original method of Quick et al., with minor modifications (Biggs, 1976; pp. 663-664), has served as the basis for present-day thromboplastin reagent preparation methods. Alternative methods are also known: For example, Biggs (1976; p. 664) teaches a method involving maceration of whole brain "for about 2 minutes with warm 0.85 per cent saline in a Waring blender." This method is but a slight modification of the Quick method, notable for its omission of the acetone extraction/dehydration step. In a second example, Hvatum& Prydz (1966) teach a method involving detergent extraction of a microsomal fraction from human brain, followed by dialysis against buffered saline solution to remove the detergent, which inhibits tissue factor activity. Because of the need to prepare microsomes by high-speed centrifugation and to remove detergent by dialysis, this method is cumbersome and not amenable to the preparation of large quantities of thromboplastin required for commercial applications.
Summarizing, the current state of the art for production of a thromboplastin reagent (from human or animal tissues) suitable for use in the PT test involves (1) selection of an appropriate tissue with high thromboplastin (tissue factor) activity [e.g., rabbit, bovine, or human brain; human placenta; or rabbit or bovine lung]; (2) dehydration and partial delipidation of the minced tissue with acetone, with further drying at ambient or elevated temperature to yield an acetone powder, and reconstitution of the acetone powder in an appropriate diluent, or tissue maceration; and (3) removal of large particles by centrifugation. Thromboplastin reagent also may be formulated via modifications to steps 2 and 3 as described above.
Methods for evaluating thromboplastin reagents
Commercially available thromboplastin reagents are of two general varieties: heterologous thromboplastins such as those derived from animal tissues (e.g., rabbit brain or bovine lung), and homologous thromboplastins such as those derived from human tissues (e.g., human placenta). As reviewed by Kirkwood (Thromb. Haemostas. 49:238-244, 1983) and Hirsh et al. Chest 95:5S-11S, 1989) thromboplastin reagents vary in their ability to detect the reductions in clotting factor activity induced by oral anticoagulants. It is generally recognized that homologous thromboplastin reagents are more sensitive to these changes in clotting factor activity than their heterologous counterparts. This increased sensitivity is desirable because it (i) permits better control of anticoagulant therapy and thereby reduces the risk of abnormal bleeding due to over-anticoagulation and (ii) increases the reagent sensitivity to minor abnormalities in coagulation factors of the tissue factor pathway.
One measure of the quality or sensitivity of a thromboplastin reagent is the "International Sensitivity Index" (ISI), which is calculated from an orthogonal regression of a test thromboplastin reagent against a reference thromboplastin reagent (as reviewed by Kirkwood and Hirsh et al.). The following is an example of how such tests are performed: A reference thromboplastin reagent of known ISI is obtained. Using this reference thromboplastin reagent and the test thromboplastin reagent, a PT determination is performed on normal and coumadin-anticoagulated plasma samples. The results are plotted as the logarithms of prothrombin times of the reference thromboplastin reagent (ordinate) vs. the test thromboplastin reagent (abscissa). Orthogonal least squares regression analysis is used to determine the slope of a straight line that best fits the data. The ISI of the test thromboplastin reagent is calculated by multiplying the ISI of the reference thromboplastin reagent by the slope of the regression line.
As noted by Kirkwood, the foregoing method for calculating the ISI of a test thromboplastin reagent represents a commonly accepted approach for determining the suitability of a thromboplastin reagent for use in the PT test. Such a test was used to demonstrate the validity and usefulness of the present invention. A recent example of the application of this method is found in an article by van den Besselaar and Bertina (1991).
Thromboplastins from cultured cells
When Quick et al. described a method for producing a thromboplastin suitable for the PT test, little was known regarding the underlying mechanisms and reactions. It is now well established that the active principle in thromboplastins and the thromboplastin reagents prepared from animal tissues such as brain, lung, and placenta is a transmembrane, lipid-associated glycoprotein known as "tissue factor" or factor III (Nemerson & Bach, 1982; Spicer et al., 1987 Nemerson, 1988). It is also widely recognized (as reviewed by Nemerson & Bach, 1982, and Drake et al., 1989) that virtually all extravascular tissues (i.e., all tissues except endothelial cells or blood cells) contain tissue factor. Furthermore, (as reviewed by Nemerson and Bach, 1982), it is known that when normal or transformed cells from extravascular tissues are grown in culture, tissue factor/thromboplastin activity also may be expressed, and that endothelial cells or white blood cells can be induced in vitro to express tissue factor by a variety of chemical or biological agents.
Attention is now directed to the prior art regarding methods for preparing thromboplastin reagents from cultured cells (as distinguished from animal tissues) and the suitability of such thromboplastin reagents for use in the PT test. A review of the recent literature disclosed the following: (1) Naito et al. (1983) prepared thromboplastins from 8 lines of cultured human gastric cancer cells by a process involving "freeze thawing and sonification" to produce a cell lysate that was subsequently extracted with "physiological saline" to yield a solution rich in thromboplastic activity. A "plasma recalcification time" was used to detect tissue factor activity, and clotting times ranging from 21.1-77.4 sec. were reported. With the exception of the cell lysis procedure, this thromboplastin was prepared according to classical methods described above; however, because the normal plasma PT was not within the range of 10-14 sec, Naito et al. (1983) does not teach a method for producing a thromboplastin reagent suitable for use in the PT test.
(2) Silverberg et al. (1989) identified tissue factor in two human pancreatic cancer cell lines and prepared solutions with thromboplastic activity from "whole cells, freeze-thawed cells, microvesicles, and membrane preparations." The preparation method was neither specified nor cross-referenced in this article. Thromboplastin activity was determined with a two-stage recalcification time test, and clotting times as low as 17 sec. were reported. Because the current PT assay procedure was not used, and because the normal plasma PT was not within the range of 10-14 sec., Silverberg et al. did not teach a method for producing a thromboplastin reagent suitable for use in the PT test.
(3) Andoh et al. (1990) taught what they described as a "one-stage method" for detecting tissue factor activity in leukemic cells. In fact, it was a two-step PT test involving (i) incubation of a leukemic cell homogenate with human plasma for 3 min. and, thereafter, (ii) addition of CaCl.sub.2 to initiate coagulation. Normal plasma clotting times equal to or greater than 54 sec. were reported. Because the normal plasma PT was not within the range of 10-14 sec, Andoh et al. did not teach a method for producing a thromboplastin reagent suitable for use in the PT test.
(4) Rehemtulla et al. (1991) transfected the gene for human tissue factor into a rodent cell line, and demonstrated that the expressed recombinant human tissue factor protein exhibited physicochemical and biologic properties similar to that of its native human counterpart. A method for preparing a thromboplastin reagent was described: "Cell suspensions at 1.times.10.sup.6 cells/ml in TBS [20 mM Tris, 150 mM NaCl, pH 7.4] were frozen in a dry ice/ethanol bath and thawed at 37.degree. C. three times, and 0.1 ml of this lysate was used in a standard one-stage clotting assay to determine procoagulant activity." Without specifying the actual PT method used, the authors stated that thromboplastins produced from these freeze/thaw lysates of recombinant cells were evaluated according to two different two-step PT methods (Levy & Edgington, 1980; Tsao et al., 1984). In this example, normal plasma clotting times in the 10-14 second range indeed were obtained. However, since the assay protocols contained significant deviations from the commonly accepted procedure for the PT test it is impossible to determine whether a suitable thromboplastin reagent was produced using the freeze/thaw method. Furthermore, the raw material for the thromboplastin reagent in the example by Rehemtulla et al. was produced from recombinant rodent cells that were expressing supernormal amounts of human tissue factor. Thus, it is impossible to determine from this example of the prior art whether the thromboplastin preparation method would be appropriate for cultured non-recombinant cells, the subject of the present invention. We prepared thromboplastin reagent from cultured non-recombinant cells by the method of Rehemtulla et al. The results of this experiment, reported in Table 1, clearly indicate that this method does not yield a satisfactory reagent.